Mechanism Of Black Phosphorus Regulating Oxidative Stress-Inflammation Cascade In Retarding Intervertebral Disc Degeneration

OBJECTIVE:To treat intervertebral disc degeneration by targeting oxidative stress with the antioxidation of black phosphorus quantum dots.The effect of black phosphorus quantum dots on anti-oxidation pathway Nrf2/ARE and local inflammation was further explored through in vivo and in vitro experiments.METHODS:Black pnosphorus quantum dots were prepared by ultrasonic liquidphase stripping method,and the materials of black phosphorus quantum dots were characterized.The biocompatibility of black phosphorus quantum dots was verified by CCK-8 and living/dead staining in vitro.The effects of different concentrations of hydrogen peroxide on Nrf2/ARE pathway in nucleus pulposus cells were investigated by qRT-PCR.The effect of black phosphorus quantum dots on Nrf2/ARE pathway in nucleus pulposus cells under oxidative stress and the relationship between Nrf2/ARE pathway and inflammation were explored by Western-blot experiment.In the animal experiments in vivo,SD rats were utilized to establish the model of intervertebral disc degeneration,and SD rats were divided into sham operation group,black phosphorus intervention group and puncture group.After corresponding intervention,the effect of black phosphorus quantum dots on antioxidation and delaying intervertebral disc degeneration was further verified by X-ray,T2-weighted magnetic resonance imaging and intervertebral disc tissue sections.RESULTS:Under transmission electron microscope,the black phosphorus quantum dots are uniformly dispersed and the morphology of black phosphorus quantum dots are regular.The average particle size of black phosphorus quantum dots measured by particle size analyzer is 8nm.The representative peaks of Raman scattering spectra of black phosphorus quantum dots are out-of-plane phonon mode(Alg)358.4cm-1,in-plane modes(B2g)438.2cm-1 and(A2g)467.1cm-1,respectively,which are consistent with the typical Raman spectra of black phosphorus.Live/dead staining and CCK-8 experiments showed that black phosphorus quantum dots had outstanding biocompatibility,and black phosphorus quantum dots could protect the viability of nucleus pulposus cells under oxidative stress.Intracellular reactive oxygen probe and JC1 fluorescence probe confirmed that black phosphorus quantum dots could clear the nucleus pulposus cells caused by hydrogen peroxide intervention and restore the mitochondrial membrane potential of nucleus pulposus cells.The results of qRT-PCR and Western-blot experiments showed that hydrogen peroxide at pathological concentration inhibited the Nrf2/ARE pathway,and the intervention of black phosphorus quantum dots could restore the viability of Nrf2/ARE pathway.The inflammation induced by oxidative stress can be alleviated by black phosphorus quantum dots,but ML385,a specific inhibitor of Nrf2,can cancel this effect,indicating that the inhibition of black phosphorus quantum dots on the inflammation of nucleus pulposus cells may be achieved by restoring the Nrf2/ARE pathway.The results of experiments in vivo showed that the height of intervertebral disk,MRI score,histological staining and immunohistochemical staining in the black phosphorus intervention group were better than those in the puncture group,which further proved that black phosphorus quantum dots could play an antioxidant role and delay intervertebral disc degeneration in vivo.CONCLUSION:Black phosphorus quantum dots have outstanding biocompatibility and antioxidation.It can reduce the oxidative stress of intervertebral disc by scavenging ROS in nucleus pulposus cells and restoring the activity of Nrf2/ARE pathway,inhibit aseptic inflammation of intervertebral disc and catabolism of extracellular matrix,and finally delay intervertebral disc degeneration in rats.This study provides a new strategy for treating intervertebral disc degeneration by targeting oxidative stress.